The present invention relates in general to a method and apparatus for controlling liquid flow in capillary electrophoresis (CE).
CE is a known technique for effecting separation of a mixture of analytes in which a voltage is applied across a capillary containing a sample, and the resulting electric field causes electrophoretic flow of charged molecular species in the sample. This technique can be utilized, for example, to separate proteins having different charges because the applied electric field will cause the differently charged proteins to travel at different velocities, thereby causing separation of them along the length of the capillary.
The charged ions have an electrophoretic mobility which is proportional to the magnitude of their charge density, and this is one of the major forces for causing separations in CE. Another force results from the ionization of silanol groups along the wall of the capillary. In particular, when a fused silica capillary is employed and is filled with an aqueous solution having a pH above approximately 2, the surface silanol groups will become ionized, resulting in a negative charge on the wall of the capillary. Cations (positively charged ions) of the aqueous solution are attracted to the negatively charged capillary wall thereby forming an electrical double layer at the capillary wall solute interface. When a voltage is applied across the capillary, these cations flow towards a cathode end of the capillary, thereby resulting in a bulk flow of fluid in this direction. This bulk flow is referred to as electroendosmosis or electro-osmotic flow, otherwise known as EOF.
Electrophoretic mobility and EOF are therefore the two major electrical forces in CE. This can be illustrated by assuming the typical situation where the EOF is greater than the electrophoretic mobility of the materials in the sample to be analyzed. Cations are electrophoretically mobilized toward the cathode end of the capillary, and their electrophoretic mobility towards the cathode is enhanced by the EOF. On the other hand, anions (negatively charged ions) are electrophoretically attracted toward the anode end of the capillary, but since the EOF is greater than the electrophoretic mobility, the anions net movement is toward the cathode with the bulk flow. In this situation, the order of flow of analytes passing a detector positioned at the cathode end of the capillary will be cations, then neutral species, then anions. It will be understood that the higher the magnitude of the EOF relative to the electrophoretic mobility, the more close together the various analytes will be as they pass the detector. Thus, if the effects of the EOF can be reduced, the analytes can be more spread out, thereby increasing the resolution of the separation.
A number of problems are presented in conducting separations with CE. First, in order to effect a separation of a mixture of analytes, some method must be employed to load the analyte mixture sample into the capillary. The most common methods are electrokinetic and hydrodynamic sample loading. Electrokinetic sample loading, also called electrokinetic injection or electromigration, utilizes both electrophoretic and electroosmotic flow to introduce a sample into the capillary. The inlet end of the capillary and a power supply's anode are placed into a sample containing vial, and a voltage is applied across the capillary for a period of time. The strength and duration of the resulting electric field ideally determine the amount of the sample introduced into the capillary; however, there is a bias in this method of sample introduction. Briefly, cations are introduced into the capillary by virtue of both electrophoretic mobility and EOF. However, uncharged species and anions are introduced into the capillary by virtue of only EOF since their electrophoretic mobility is either zero in the case of uncharged species, or in the direction of the anode in the case of anions. Thus, anions migrate into the capillary more slowly than uncharged species because of the electrical attraction of the anions toward the anode. Therefore, the different electrophoretic mobilities of the analytes arising from their different charge densities is a source of bias in sample loading by electrokinetic injection. Another bias occurs because different electrolyte buffer solutions have different electrophoretic and EOF rates, resulting in different amounts of sample being injected.
Several approaches to eliminating these sources of bias in electrokinetic injection have been reported, all of which implicitly aim to decouple electrophoretic flow from EOF, thereby accomplishing sample introduction through EOF alone. This decoupling has in all cases been achieved through approaches involving alterations to the capillary structure itself, such as by introducing porous glass or a frit, coupling to an additional capillary or fracturing the capillary. These approaches all require specialized capillary manufacturing techniques, are rather complex and labor intensive, and do not address the problems associated with the changes in the electrolyte buffer solution.
A nonelectrical sample loading method, such as hydrodynamic sample loading, avoids all of the problems associated with electrokinetic sample loading. All hydrodynamic sample loading methods involve, by one means or another, a pressure differential between the inlet and outlet ends of the capillary. This can be accomplished simply by raising the inlet end of the capillary above the outlet end, or through the use of either a pressure pump or syringe pump at the inlet end of the capillary, or a vacuum pump at the outlet of the capillary. Most known hydrodynamic sample loading methods are rather bulky and expensive to implement. Further, all of these methods suffer from potential band broadening with an attendant loss of resolution if the pressure differential is so large, or the inside diameter of the capillary is so small, that the injection front is distorted. For example, in one experiment with an optimized commercial CE system using pressures as low as 0.497 psi for sample loading, the experimentally determined injected volume per unit time deviated from a calculated theoretical value by 6.1%.
Another problem with conventional CE systems is that of controlling the effects of the EOF. Because the EOF is a source of a zone broadening in free zone CE, or disturbs focused zones in isoelectric focusing CE, many investigators have attempted to eliminate EOF entirely. Reducing the EOF below zero, i.e., reversing the direction of the EOF, or increasing the EOF may be advantageous in some applications such as micellar electrokinetic capillary chromatography (MECC) because resolution may be increased and analysis time reduced.
A number of techniques have been employed to increase, decrease or eliminate the effects of the EOF. One such technique involves coating the inner surface of the capillary with a material whose charge is different from that of the uncoated capillary. Use of an electrically neutral coating material would eliminate the surface charge that gives rise to EOF, while use of the material whose charge is more negative than that of the inner surface of the capillary would increase the EOF. On the other hand, use of a material whose charge is positive would reverse the direction of the EOF. However, the use of coatings to change the EOF has drawbacks. In particular, the coatings degrade with use over time and suffer from the complexity involved in the coating procedures. Also, these methods all result in a capillary with a changed, but not adjustable EOF.
Another technique for changing the EOF involves the use of electrolyte buffer additives which cause Coulombic repulsion between the capillary surface and the analytes to reduce the EOF. Other additives may be used to increase or reverse the EOF. Unfortunately, use of such additives presents the possibility that the additive may adversely affect the material to be analyzed. Further, the use of these methods once again does not provide for adjustment of the EOF during CE. Yet another method for changing or controlling the EOF involves the manipulation of the bulk flow through the application of electric fields or temperature gradients, although these methods tend to be overly complex.
A third problem in conventional CE systems is presented by the need to move the sample material past a detector of some kind after the analytes therein have been separated. More particularly, once the individual components in the analyte mixture sample have been separated, they are caused to flow past a detector, such as a UV absorbance, radioactive decay or fluorescence detector, so that some attribute of the component can be sensed thereby. Then the component flows out of the capillary into the reservoir disposed at the outlet end thereof. Electrophoretic mobility, EOF and pressure have all been utilized to achieve this mobilization through the capillary. The same problems associated with sample loading occur in mobilization; namely, electrophoretic mobilization may be biased, EOF is difficult to control and pressure systems have been relatively crude and inaccurate. Once again, none of these approaches allows the real-time control of the rate of mobilization.
An example of the mobilization problem occurs in isoelectric focusing (IEF) CE. In IEF CE, a pH gradient is formed along the length of the capillary and analytes migrate through this gradient until they reach the pH zone where their net charge is zero, and they stop moving. Thus, the EOF needs to be eliminated so that there is no bulk fluid flow. In this manner, an analyte is focused into a zone at its isoelectric point. After the separation is completed in this manner, the zones must be mobilized past a detector, ideally without any disturbance of the focused zones.
For this purpose, an electrophoretic mobilization technique called salt mobilization has been developed and commercialized. Once focusing is completed, the electric field is turned off, salt is added either to the anode or cathode buffer reservoirs and the field is reapplied, thus causing an excess of either H.sup.+ or OH.sup.- to enter the capillary thereby changing the pH gradient and causing migration toward the cathode or anode as the case may be. This system is ideally set up so that the pH gradient will flow past the detector window on its way toward the electrode. This procedure suffers from the need to perform the multiple steps discussed above. In addition, the focused zones are susceptible to diffusion while the field is turned off and to an electrophoretic bias during the electrically driven mobilization. Hence, reproducibility and resolution are often adversely affected.
Another technique used for mobilization in IEF CE is to sharply reduce, but not eliminate, the EOF, such as through the inclusion of methyl cellulose in the electrolyte buffer. The reduced EOF can then be employed for mobilization once the separation is completed, however, this technique requires a compromise between the separation resolution and the mobilization speed. Further, the rate of mobilization cannot be adjusted with this technique.